MUC1 mucin expression as a marker of progression and metastasis of human colorectal carcinoma

GASTROENTEROLOGY 1994;106:363-361

MUCl Mucin Expression as a Marker of Progression and Metastasis of Human Colorectal Carcinoma SHOJI

NAKAMORI,”

and TATSURO

DAVID

M. OTA,’ KAREN

R. CLEARY,§

KEIRO SHIROTANI,”

IRIMURA*,II

Departments of *Tumor Biology, ‘General Surgery, and §Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and l’Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences. The University of Tokyo, Tokyo, Japan

Background/Aims: The MUCl mucin distributes among a variety of epithelial tissues (except the intestinal epithelia) and is often detectable in colorectal carcinoma tissues and cell lines. This study aimed to elucidate whether MUCl mucin expression correlated to the progression of colorectal carcinomas. Metho&: We collected 113 tissue specimens, including primary colorecta1 carcinoma, normal mucosa, liver metastases, lymph node metastases, and normal livers from 58 patients with colorectal carcinoma. Immunohistochemical staining and Western blotting analysis with mature MUCl mucin-specific monoclonal antibodies were performed. Results: The levels of mature MUCl mucins were signiflcantly higher in carcinoma tissues than those in normal colonic mucosa (P < 0.001). Futthermore, the levels of mature MUCl mucins were significantly higher in primary tumors from patients having metastasis or metastatic tumors than in primary tumors from patients without metastasis (P < 0.001). In the primary sites, mature MUCl mucin levels apparently increased according to progression of the stages (P c: 0.001). Conc/usions: These results strongly suggest that mature MUCl mucins become ectopically expressed in colorectal carcinoma progressed to the metastatic stages and that mature MUCl mucins may be a useful marker for advanced colorectal carcinoma.

he presence or absente of metastasis is the most critical factor in determining the prognosis of paT tients with colorectal cancer.’ Sensitive diagnostic methods to identify a group of patients at high risk of metastases and recurrence after surgical resection of the primary tumors should provide useful information for medical oncologists. Identification of specific cellular and biochemical markers that change as early colorectal carcinoma progresses to metastatic tumors should be developed for this purpose. Recent studies with experimental animals from many laboratories, including ours, have shown that highly metastatic tumor cells possess a variety of specifïc cel1 surface properties that are different from poorly metastatic tumor cells.z-5 It is widely accepted thal: the structure and distribution of cel1 surface glycoproteins changes during malignant transformation and

tumor

progression.

These

changes

are also believed

to

influence the metastatic behavior. I;or example, altered expression of glycoproteins on the cel1 surface are frequently found to be associated with altered adhesion,“,7 invasion,s.8 and immunogenicity nition lectively cells.2-4 We changes

influence

or other immune

’ Perhaps

phenomena.“-’

the

are interested

these

metastatic

recog-

phenotypes

potential

in determining

col-

of tumor

the biochemical

of cel1 surfaces and extracellular

molecules

dur-

ing cancer progression that influence the metastatic potential of human colorectal carcinoma. Using clinical tumor specimens, we previously showed that the levels of several different high molecular weight glycoproteins (mucins) human

consistently colorectal

high molecular a combination

increased

carcinoma weight

or decreased

tissues.”

sialoglycoproteins

of polyacrylamide

in advanced

-” The changes

in

were shown by

gel electrophoresis

and

wheat germ agglutinin-binding.“,‘7 In these previous studies, the molecular nature of the sialoglycoproteins was not clear. In the present study, we have focused on the expression of MUCl mucin in colorectal carcinomas, paying

special attention

to its relationship

to the stage

of the disease. The MUCl gene encodes core polypeptides corresponding to highly glycosyla.ted, high molecular weight glycoproteins synthesized i.n normal and malignant mammary epithelial tissues.‘Hm2’ The complementary DNA (cDNA) coding MUCl core protein was previously isolated from human mammary and pancreatic carcinoma cells.“~‘” Immunohistochemical studies by Zotter et al. have shown that MUCl is present on the surface of a variety

of human

normal

and neoplastic

tis-

sues, whereas the large and smal1 intestines have been immunohistochemically negative for the binding of MUCl mucin-specific monoclonal antibodies (MAbs).” Recently, the organ-specific expression of MUCl mucin Abbreviations used in this paper: DPBS, Dulbecco’s phosphate buffered saline; MAb, monoclonal antibody; M,, relative molecular weight; SDS, sodium dodecyl sulfate. 0 1994 by the American Gastroenterological Association 001~5085/94/$3.00

354

GASTROENTEROLOGY Vol. 106, No. 2

NAKAMORI ET AL.

A 12345678

9

1011

Kd

6

880

50 r-

0

20

40

60

100

80

amount of protein (CcCl) Figure 1. Quantitation of mature MUCl mucins by the binding of anti-MUCl mucin MAb HMFGl. (A) Autoradiogram following 1251-labeledgoat anti-mouse immunoglobulin G antibody binding to different amounts of Capan-1 cell extracts (lanes 2-6) and a colorectal carcinoma tissue extract (lanes 7-11) electrophoretically separated and reacted withMAb HMFGl. Molecular weight markers used are intact laminin (M,, 880,000), large subunit of laminin (& 440,00O),small subunit of laminin (A$, 220,000), and &galactosidase (M,, 120,000). Arrowheads indicate the position of the MUCI mucins. (8) The radioactivity associated with mature MUCl mucins in the gels were counted and plotted according to the amount of protein loaded in each lane. 0, Capan-1 cel1 extracts; & colorectal carcinoma tissue extracts.

was clearly some

shown

colorectal

were shown whether

carcinoma

to express

these

colorectal

ectopically

In this

report,

of MUCl

mucin

colorectal

carcinoma

nostaining

method.

of highly

glycosylated

by an MAb,

HMFG1,‘4

of colorectal

carcinoma.

examined

in a large with

results

number a direct

have MUCl

correlated

in

role in metastatic

we have

specimens

in cultures mucins

indicated mucins

the levels

(Bl,

size in 56 primary tumors was 4.7 cm (range, l.O- 12.5 cm). The mean tumor size according to the stage was A, 5.3 cm (range, 1.0-12.5 cm); B, 3.5 cm (range, 1.5-6.0 cm); C. 5.2 cm (range, 3.0-8.5

of human

Tumor

the

identified

to the clinical

stage

cm); and D, 5.2 cm (range, 2.5-8.0

cm).

Tissue Specimens and Protein Extraction

gel immuthat

The

of primary tumors according to stage was A, 7; B, 17 11; B2, 6); C, 16 (Cl, 6; C2, 10); and D, 16. Mean tumor

number

It was unclear

MUCl

a biological

expression The

grown

mucins.‘““o

expressed

play

rent liver tumors), and 3 poorly differentiated adenocarcinoma.

mice. *s Interestingly,

cel1 lines

MUCl

carcinoma

phenotype.

levels

in transgenic

specimens

of approximately

0.5- 1.0 g were

obtained from the intraluminal edges of colorectal tumors. Normal mucosa from an area approximately 5-cm from the primary carcinoma and metastatic tumor and adjacent normal

Materials and Methods

tissues in liver or lymph node was obtained when available. Necrotic and ulcerative portions of the tissues were excluded. Normal colonic mucosa was dissociated from muscle and con-

Patients and Clinicopathological Information

nective tissues. Al1 tissues were frozen as soon as possible after resection and stored at -70°C until use. Frozen tissues were

Fifty-eight patients who had undergone surgical resections of adenccarcinoma of colon and rectum and hepatic metastasis at the University of Texas M. D. Anderson Cancer Center were selected for this study. Age, sex, tumor kation, size, and pathological

histology,

tumor

data such as depth of invasion, presence of

lymph node metastasis, and presence of hepatic metastasis were obtained from the hospital records of these patients. The staging was based on the Dukes’ classification’ with slight modihcations as previously described.‘“-‘* This study cohort consisted of 30 men and 28 women (mean age, 61 years; range, 40-85 years). The location of 56 primary tumors was 19 in the proximal colon (cecum, 6; ascending colon, 7; and transverse colon, 6) and 37 in the distal colon (descending colon, 6; sigmoid colon, 13; and rectum, 18). Histologically, there were 17 well-differentiated adenocarcinoma, 36 moderately differentiated

adenocarcinoma (including two recur-

minced

into smal1 pieces and extracted

with 1 mL of 5 mmol/

L Tris-HCl buffer (pH 7.4) containing 0.25 mol/L sucrose, 0.05 mmol/L CaCl?, 0.001 mmol/L phenylmethylsulfonylfluoride, and 0.5% Nonidet P-40 at 4°C for 6 hours. Nonidet P40 (Sigma Chemical Co., St. Louis, MO) was used to solubilize MUCl mucins because MUCl mucins had a transmembrane domain and associated with membrane by this domain. Supernatant fraction was collected by centrifugation at 13,000g for 5 minutes. Protein content of tissue extracts was determined by the method of Lowry” and normalized at 3 mg/mL with the same buffer used for the extraction of tissues.

Polyacrylamide Gel Electrophoresis and Immunostaining The tissue extracts were mixed with a half volume of 187.5 mmol/L Tris-HCl containing 5 % sodium dodecyl sulface

February 1994

MUCl

EXPRESSION IN COLORECTAL CARCINOMA

355

buffer) for 4 hours at 22°C and then soaked in 20 mL ‘?goat anti-mouse d

T T TT

TT

T T T

NTNTN

T NT

NT

NTH

immunoglobulin

ICN Radiochemicals,

G antibody (2 X 106 cpm/mL;

Irvine, CA) in 0.25 mol/L Tris-HCl

(pH 7.4) containing

buffer

0.13 moliL NaCl and 0.05% Tween-20

for

4 hours at 22°C. The gels were washed repeatedly with washing buffer until the radioactivity

of the wash became lower than 100

cpmiml,

dried under vacuum, and then exposed to Kodak KAR-

5 films

(Eastman

Kodak,

Rochester,

screens at -7O’C. Underglycosylated were identified supernatant

by the binding

B

LNT

L NTH

NTH

880,000

mucins culture

separated tissue extracts with MAb SM3 used for MAb HMFGl.

Estimation of Tissue Contents of Mature MUCl

440,000

To quantitate

the levels of mature MUCl

tions of gels (1.5-cm as judged

was counted

described.“.” Figure 2. Profiles of direct immunostaining of tissue extracts from normal and malignant colorectal tissues electrophoretically separated and reacted with MAb HMFGl. Bound antibodies were detected by 1251-goatanti-mouse immunoglobulin G followed by autoradiography. Autoradiograms (as shown in this figure) were used to localize the position of mature MUCl mucins. On the top of gels are the stages. T, primaty tufYtOrS; N, nOrITEd mucosa; H, her rrWtaStaSt?S; L, lymph node metastases; NL, normal liver. The nine let? lanes of A contain primary tumors obtained from nine different patients. The 13 right lanes of A and all the lanes of B contain normal mucosas, primary tumors, lymph node metastases, liver metastase% or a normal liver tissue. Samples obtained from the same patient were bracketed in these latter cases. Molecular weight markers are the same as in Figure 1.

long) corresponding

from an autoradiogram

radioactivity ular weight

were cut, and the associated counter

corresponded

to the relative molec-

(M,) range between

400,000

as previously

and l,OOO,OOO. The

based on the radioactivity

mucin in the extracts (10 pg protein) creatie carcinoma

with MUCl

of Capan-1

cells, which was included

tetraa.cetic minures. subjected

acid, and 30% glycerol

and heated at 100°C for 5

A 25-~1 sample corresponding to SDS-polyacrylamide

polyacrylamide

under

to 50 pg prorein

gel electrophoresis

gels as previously

sis was performed

ethylenediamine-

described.”

constant

was

in 5%~

-” Electrophore-

current

(25 mA/l.S-cm

LO-cm wide slab gel) for 2 hours. After electrophoresis,

the gels were stained

with

Coomassie

brilliant

blue

(Sigma; 0.25 g/L in 10% acetic acid and 25% isopropyl hol) and destained alcohol. Tris-HCI

with

R-250 alco-

10% acetic acid and 10% isopropyl

The gels were washed

repeatedly

buffer (pH 7.4) containing

with

25 mmol/L

0.13 mol/L NaCl for 4

human pan-

in each gel.

Isolation and Analysis of RNA prepared

Poly( messenger RNA (mRN.A) of tissue sample was with the FastTrack mRNA isolation kit (Invitrogen,

San Diego, CA). For Northern mRNA

blotting

per sample was denatured

analysis, 5 pg of poly(

by hearing

minutes in 50% (vol/vol) formaldehyde,

at 65°C for 15

subjected

resis on 1%’ agarose 2.2 mol/L formaldehyde transferred to nylon membranes

(SDS)l, 3% 2-mercaptoethanol, 1.5 mmol/L

porband

on a gamma

This portion

count was normalized

mucins,

to the MUCl

to electrophogel,‘? and then

(Hybond N; Amersham,

ton Heights, IL) as suggested by the manufacturers.

thick,

intensifying

of MAb SM3 (hybridoma

was performed under the same condition

NTHNTHNLNT

with

provided by Dr. Sandra J. Gendler).32 Immunostain-

ing of electrophoretically NT

NY)

precursors of MUCl

was performed

Arling-

Hybridization

at 42’C for 16 hours in 6X SSC (1 X SSC is 0.15

mol/L NaCl and 15 mmol/L sodium citrate), 1% glycine, 0.1% polyvinylpyrolidone, (Pharmacia,

Uppsala,

0.1% bovine serum albumin, Sweden), 50% formamide

0.1% Ficoll

(volivol) in the

presence of 100 PgimL transfer RNA from Escbericbiu coli (Sigma), and “P-labeled

MUCl

cDNA

probes’* (pMUC10

was a gift

from Dr. Sandra J. Gendler) for 16 hours. The filters were then washed twice for 10 minutes each in 2 X SSC solution with 0.1% SDS, followed by additional

washes using 0.2X SSC and 0.1%

SDS at 45°C for 1 hour. Finally, they were exposed to Kodak KAR-5

films with intensifying

screens at -7O’C.

the equality of the amount of poly(

mRNA,

To examine

the nylon mem-

MAb HMFGl (provided by Dr. Sandra J. Gendler, Mayo Clinic, Scottsdale, AZ)‘“.‘” was used. This antibody recognizes

branes were washed in 0.05% SDS at 90°C for 30 minutes and developed to check complete removal of previous probes, then we rehybridized the membrane with p-actin probes using the

highly glycosylated

same procedure

hours at 22°C. For the quantitation

(mature)

MUCl

of mature

MUCl

mucin,

mucins.“’ The neutralized

gels were soaked in 20 mL of MAb HMFGl (hybridoma culture supernatants) diluted 1:lO in 25 mmol/L Tris-HCl buffer

0.13 mol/L NaCI and 0.1%

Tween-20

(washing

above.

Immunohistochemical Distribution of Mature MUCl Mucins in Colorectal Carcinoma

(pH 7.4) containing 0.13 mol/L NaCl, 0.05% Tween-20, and 3% hovine serum albumin for 16 hours at 4°C. The gels were washed repeatedly with 25 mmol/L Tris-HCl buffer (pH 7.4) containing

as described

mucins

Immunohistochemical was examined with

localization of mature MUCl 12 primary adenocarcinomas and

356

GASTROENTEROLOGY Vol. 106, No. 2

NAKAMORI ET AL.

Table1. Summary

of Mature

MUCI

Variable Age (mean, 61 years) <61 261 Sex Male Female Location Proximal (to splenic flexure) Distal Size (mean, 4.7 cm) 54.7 cm >4.7 cm Histological differentiation Well Moderately and poorly Type of tissues Normal mucosa Normal liver Primary tumor Metastatic turno? Stage Stage A Stage B Stage C Stage D Presence of metastasis Without metastasis With metastasis

Mucin

in 113 Tissues

Levels

Derived

From 58 Patients

With Colorectal

Carcinoma

No.

Relative intensity# (mean + SD)

27 31

0.31 f 0.26 0.28 f 0.26

<61 vs. ~61

NS

30 28

0.33 + 0.29 0.26 5 0.20

Male vs. female

NS

19 37

0.38 t 0.31 0.24 t 0.22

Proximal vs. distal

NS

31 25

0.23 2 0.23 0.38 2 0.31

54.7

17 39

0.28 2 0.43 0.33 5 0.30

Well vs. moderately and poorly

NS

34 3 56 20

0.10 + 0.05 0.05 5 0.10

Normal mucosa vs. normal liver

NS

7 17

0.09 _f 0.04 0.17 + 0.10

16 16

0.34 2 0.28 0.46 2 0.27

24 32

0.15 2 0.09 0.40 + 0.27

Probability

Differente

0.70 + 0.72

cm vs. >4.7

NS

cm

Normal mucosa < primary tumors Normal mucosa < metastatic tumors Primaty tumors < metastatic tumors

P < 0.001 P < 0.001 P < 0.001

Normal mucosa vs. stage A Normal mucosa < stage B Stage A < stage B Stage B < stage C Stage B i stage D

P P P P

Without < with

P < 0.001

< < < <

NS 0.05 0.05 0.05 0.001

NOTE. Determined by MAb HMFGI binding to electrophoretically separated tissue extracts. NS, not significant (P > 0.05). “Antibody binding to electrophoretically separated tissue extracts normalized to standard extract. bFifteen liver metastases and five lymph node metastases.

three liver metastases. Sections (4+m) were prepared from formalin-fixed and paraffin-embedded permanent pathological

tests for nonparametric

specimens. The sections were deparaffinized and treated with 0.03% hydrogen peroxide in methanol for 30 minutes to black endogenous peroxidase activity. The sections were rehydrated and washed with Dulbecco’s phosphate-buffered saline (DPBS) and then incubated with 1% bovine serum albumin dissolved in DPBS overnight at 4°C. The sections were then incubated with MAb HMFGl

(hybridoma

1: 10 in DPBS containing

culture

1% bovine

supernatant

serum

albumin)

diluted for 2

hours at room temperature. The sections were washed several times with DPBS and treated with biotinylated goat antimouse immunoglobulin G (Vector, Burlingame, CA) for 1 hour. After repeated washing with DPBS, sections were further incubated with solution of avidin-biotinylated peroxidase complex (Vector). A mixture of 1 mL of 0.4% S-amino-l-ethylcarbazole dissolved in N,N’-dimethylformamide, 100 mL of 20 mmol/L sodium acetate (pH 5.2), and 70 /.tL of 30% hydrogen peroxide was used as a peroxidase substrate. The sections were mounted with gelatin-glycerin mounting agent.

Statistical Analysis Statistical

test or Welch’s

analyses

were performed using Student’s data, Kruskal-Wallis

t test for parametric

t

data,“’ and simple

regression

analysis

tests.”

Results Quantitation of Mature MUCl Mucins adjacent

After extraction of colorectal carcinoma tissues or normal mucosa tissues with 5 mmol/L Tris-

HCI buffer (pH 7.4) containing

0.25 mol/L sucrose, 0.05

mmol/L CaCl>, 0.001 mmol/L phenylmethylsulfonylfluoride, and 0.5% Nonidet P-40, >90% of substances reactive with MAb HMFGl were solubilized; the residual pellets did not contain a significant percentage of MAb HMFGI-reactive substances. To determine the range of linearity in antibody binding to electrophoretically separated MUCl mucins reactive with MAb HMFGl, serially diluted Capan-1 cel1 extracts and serially diluted extracts of a primary tumor tissue sample were loaded on gels, separated by electrophoresis, and immunostained with 1251 anti-mouse immunoglobulin G. As shown in Figure 1, bound radioactivity was proportional to the amount of Capan-1 cel1 extracts (0-20 pg

MUCl

February 1994

between

0

1.6

the levels of mature

sex, tumor 0

0 0

4 ..<. -& ..i

Wallis

-

_

T D

tissues.

tissues

shows a scattergram

D metastasis Flgure 3. Scattergram between stages of the disease and mature MUCl mucin levels presented by relative intensity. The analysis is based on the results from 56 primary colorectal carcinoma tissues. The number of specimens analyzed for each categov are indicated.

and age, or tumor

between

that there is a significant

rank

< Dukes’ A, Dukes’ B < Dukes’ tumors.

The H value acFigure

of the data from 56 primary

In this figure, the relative

ized to standard

nor-

of the Kruskal-

to this test was 54.0 (P < O.OOl).”

specimens.

357

was seen in the

expression

The results

C < Dukes’ D < metastatic cording

mucins

classification,

differente

mucin

test also indicated

order: normal

u

i..

MUCl

mal and carcinoma

MUCl

histological

a significant

levels of mature

E

0

location,

size. However,

8

0

EXPRESSION IN COLORECTAL CARCINOMA

extract

tumor

tissues was plotted

simple

regression

of mature against

(P < 0.001

intensity MUCl

3

tumor normal-

mucins

in

the stages. Significant

in the F test)‘* was found

between

stages and MAb HMFGl binding. The results of comparisons (Student’s t tests or Welch’s t tests) between

different stages are also summarized in Table 1. The levels of mature MUCl mucins identified by the binding protein)

and tumor

from the specificity cosylated

MUCl

tion of MAb polyacrylamide

sample (0- 100 pg protein). of MAb HMFGl

toward highly

mucins** and the relatively

of MAb HMFGl

Judging gly-

slow migra-

HMFGl-reactive substances after SDSgel electrophoresis, this procedure is con-

sidered

to be quantitative

mature

MUCl

mucins.

to determine

the amount

of

We chose to use 10 l.tg of Capan-

stages than

comparison

was performed

MUCl adjacent Table

The presence of mature

MUCl

from 113 tissues from 58 patients

mucins

in extracts

with colorectal

noma were analyzed by direct gel immunostaining MAb HMFGl Typical profiles of immunostaining

carciusing from

22 patients with colorectal carcinoma were shown in Figure 2. Radioactivity was seen associated with components that migrated to areas corresponding to molecular

Another

based on the ratio of mature

in the primary

mucosas

colon carcinomas

those at early stages. tumors

from the same patients.

to those in the The results

2 show that the values were significantly

in

higher

stages.

Comparison of the Levels of Underglycosylated MUCl Mucin Core Polypeptides and Poly(A mRNA for MUCl Mucin Core Polypeptides

on gels loaded with tissue extracts.

Direct Immunostaining with Anti-MUCl MAb HMFGl in Specimens of Normal Colonic Mucosa, Colorectal Carcinomas, and Metastatic Tumors

mucins

at advanced

1 cel1 extract as an internal standard in each gel to normalize the radioactivity associated with mature MUCl mucins

was higher in primary

at advanced

mucin

To determine whether increased mature MUCl in the advanced colorectal carcinoma was caused

by increased

MUCl

mucin

core polypeptides,

the levels

of underglycosylated MUCl mucin precursors were determined by the use of MAb SM3 in a gel staining experiment. With these selected specimens, we also compared the levels of poly( mRNA for MUCl mucin core polypeptides. The results with fïve sets of specimens (normal mucosas and primary carcinomas) are shown in Figure 4. A high leve1 of mature MUC1 mucins reactive

weights >400,000. In a few instances, two major bands apparently correspond to length polymorphism caused by a variable number of tandem repeats in the MUCl whereas in other instances, a high molecular gene,” weight broad band (larger than 400,000) was observed. As shown in Figure 2, intensity of the bands varied from one sample to another. Extracts of carcinoma tissues contained a much greater quantity of mature MUCl mucins than normal tissues. In normal tissues, intensity of antibody binding was very weak. The relative intensity normalized to standard extract derived from analysis of 113 specimens are summarized according to clinicopathological features in Table 1. There was no correlation

Table 2. Ratio of Mature MUCl Mucin Levels in Primary Tumors to Corresponding Normal Mucosas From the Same Patients as Classified by the Stages Stage Tumor/normal 52.0 >2.0 25.0

ratio

and ~5.0

Total aKuruskal-Wallis

A

B

C

D

6 0 0

5 1 0

2 6 1

2 5 6

15 12 7

6

6

9

12

34

test: H = 11.08,

P = 0.0113.

Totala

358

NAKAMORI

GASTROENTEROLOGY

ET AL.

Vol. 106,

No. 2

AAAAA

NTNTNTNTNT

880Kd

mAb HMFGI binding

Figure 4. Comparison of the levels of mature MUCl mucins shown by the binding of MAb HMFGl (top panel) and precursor core polypeptides of MUCl mucins shown by the binding of MAb SM3 (middle panel) after electrophoretic separation of tissue lysates. T, primary tumors; N, normal mucosa. Molecular weight markers used are the same as in Figure 1. In the bottom panel, poly( mRNA for MUCl mucin core polypeptides revealed by Northern blotting analysis are shown (upper portion). The results of the Northern blotting analysis of pactin poly( mRNA after rehybridization of the same membranes were included (lower portion).

440 Kd 440 Kd

mAb SM3 binding

220 Kd MUCI

mRNA

pactin mRNA with MAb HMFGl nomas

as compared

was observed with two cases of carciwith

normal

mucosas

(cases 1 and

5). In one case (case 2), a slight increase of mature

MUCl

mucins was seen. To our surprise, the levels of MUCl mucin precursor peptides identified by the binding of MAb SM3 to carcinoma tissues of cases 2 and 5 were slightly lower than the levels in corresponding normal

at the primary

sites showed MAb reactivity

plasm. Similar staining ing liver metastases.

patterns Normal

in their cyto-

were seen in correspond-

intestinal

mucosa and nor-

mal hepatic parenchyma were not stained by this antibody, which is consistent with previous reports.18.‘7

Discussion

mucosas. Furthermore, the levels of poly( mRNA of MUCl mucin core polypeptide in carcinoma tissues in these three cases were lower than the levels in normal

Mucins are highly glycosylated, high molecular weight glycoproteins with unique core polypeptides rich in threonine, serine, and proline. The composition of the

mucosas. These results clearly indicated that increased mature MUCl mucins in advanced colorectal carcinoma tissues were not caused by increased levels of MUCl mucin core polypeptides.

oligosaccharides is variable?’ Mucins are often secreted and located on epithelial cel1 surfaces, and their primary function is thought to lubricate and protect the surface

Immunohistochemical Localization MUCl in Section of Colorectal Carcinoma Tissues

of

The immunohistochemical localization of MAb HMFGl binding to MUCl mucins expressed in colorecta1 carcinomas (a total of 12 primary carcinoma sections and four corresponding liver metastases) were examined. In some primary carcinomas, morphologically normal mucosa was present in the sections. Typical staining profiles are shown in Figure 5. In al1 cases, the intensity varied from one part to another. Prominent staining at the luminal epithelial surface was evident. Secreted intraluminal material was also stained. Some tumor cells

of epithelia. Altered glycosylation described in a variety of carcinoma

of mucins has been tissues.38m”

In this study, we used a specific MAb in combination with electrophoretic separation to identify different forms of MUCl mucins. This procedure allowed US to detect low levels of MUCl mucins in a relatively large number of colorectal carcinoma tissue specimens representing a variety of clinical stages. By using MAb HMFGl specific for heavily glycosylated forms of MUC 1 mucins and MAb SM3 specific for underglycosylated forms of MUCl mucin core polypeptides, mature and precursor MUCl mucins were separately identified. The results show that the levels of mature MUCl mucins in primary colorectal carcinomas with metastases (stages C and D) or metastatic tumors were greater than in those without metasta-

February 1994

MUCl

EXPRESSION IN COLORECTAL CARCINOMA

359

Figure 5. Immunohistochemical staining of human colon carcinoma tissue sections with MAb HMFGI. The sections were taken from surgical specimens from a patient with moderately differentiated recta1 carcinoma of stage D and a liver metastasis. (A) A section of a primary tumor. The binding of MAb HMFGl is seen at the luminal surfaces and in the secreted materials. (6) A corresponding liver metastasis. MAb HMFGl does not bind hepatocytes or other cells in the liver.

ses (stages

A and B). Therefore,

expression

seems to be related

mature to tumor

MUCl

mucin

progression

to

the advanced stages. Vogelstein et al. have shown that allelotypic changes, ras gene mutation, and changes in tumor suppressor genes occurred during the evolution of human

colorectal

carcinomas.47-45

Their studies have not

identified genetic changes involved in the progression of colon carcinoma from nonmetastatic to metastatic phenotypes.‘” We have been studying the theory that phenotypic changes are involved in tumor progression. The

the cells advantages to disseminate, invade, and survive at distant organ sites.“’ The mature MUCI mucins are likely to be one of these determinanrs that are characteristic of advanced colorectal carcinoma. Colon carcinoma cel1 lines that are metastatic in mice “La’ have not been tested for their levels of ectopic mucins, and it would be interesting to test whether selected cells for increased

mature MUCl mucins might be one of these phenotypes correlated to tumor progression to the metastatic stages

mature MUCl mucins possess hig’her metastatic ability than cells with lower mature MUCl mucins. Investigators have indicated that MUCI mucin core protein is a T cel1 epitope, which may be recognized by cytotoxic T cells, and T cel1 cytotoxicity can be inhibited

and may be a useful marker for the progression of human colorectal carcinoma. We have previously shown that colorectal carcinomas at advanced stages are phenotypically different from counterparts at early stages in the expression of cel1 surface glycoproteins, and we proposed that phenotypes associated with advanced carcinomas influence the biological behavior of metastatic colorectal carcinoma cells.‘2m’5 Metastatic tumor cells are known to express a variety of phenotypes that collectively give

by the anti-MUCl core peptide antibody.‘“,” MUCl mucins may be used as a target molecule if they are underglycosylated. Hareuveni et al, have shown that pretreatment with a recombinant vaccine virus carrying the MUCl gene can inhibit the development of tumors induced by a rus oncogene-transformed fibroblast line expressing the gene.51 Whether MUCl mucins ectopically produced by colorectal carcinoma cells are capable of inducing specific cytotoxic T cell(s is not known. The

360

NAKAMORI ET AL.

GASTROENTEROLOGY Vol. 106, No. 2

present results and our preliminary from human

results on the analysis

of MUCl

mucin

colon carcinoma

indicated

that they were heavily glycosylated.

it is not likely that underglycosylated peptides

are exposed

MUCl

cel1 lines Therefore, core poly-

on the surface of colon carcinoma

cells. The regulatory ture MUCl results

shown

transcription

basis for the ectopic expression

mucins

remains

in Figure of MUCl

of ma-

to be further elucidated. 4 clearly

mucin

show that

The

aberrant

core polypeptides

was not

likely to be the cause. The levels of MUCl

mucin precur-

sor polypeptides

of MAb SM3,

which

apparently

identifìed

mRNA,

did not correlate

mucins

detected

processing

to the levels of MUCl

to the levels of mature

by MAb

or stability

the cells should

by the binding

corresponded

HMFGl.

of MUCl

be responsible

Therefore,

MUCl altered

mucin precursors

within

for the increased

expres-

sion of mature forms of these molecules.

We have recently

found that the levels of MUCl

core polypeptide

mucin

mRNA in cultured colon carcinoma cells was modulated by conditioned media of normal colonic connective tissues.”

It is likely that such modulating

colon carcinoma

cells as wel1 as colonic

substances

affect

epithelial

cells

in vivo.

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Received January 21, 1993. Accepted August 17, 1993. Address requests for reprints to: Tatsuro Irimura, Ph.O., Oivision of Chemical Toxicology and Immunochemistory, Faculty of Pharmaceutical Science, The University of Tokyo, Hongo 7-3-1, BunkyoKu, Tokyo 113, Japan. Fax: (81) 3-38159344. Supported by the National Institutes of Health grants ROICA39314 and ROI-CA 50231; Texas Advanced Technology Program grant 1549; grants from the Ministry of Education, Science, and Culture of Japan; the Human Science Foundation of Japan; the Association for Biotechnology Research of Japan; the Terumo Foundation; and the Takeda Foundation (T.I.). Dr. Nakamori’s present address is: Department of Surgical Oncology, Center for Adult Diseases, Osaka 537, Japan. Dr. Ota’s present address is: Division of Surgical Oncology, Ellis Fischel Cancer Center, Columbia, Missouri 65203. The authors thank Drs. Sandra J. Gendler, Garth L. Nicolson, and Marsha L. Frazier for their gift of antibodies and helpful discussion on this work and Mariko Takano and Yoko Tsuchiya for their assistante in the preparation of this manuscript.